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1.  A New Method for Stranded Whole Transcriptome RNA-seq 
Methods (San Diego, Calif.)  2013;63(2):126-134.
This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
doi:10.1016/j.ymeth.2013.03.023
PMCID: PMC3739992  PMID: 23557989
RNA-seq; transcriptome; duplex-specific nuclease; gene expression1
2.  Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients 
Clinical Epigenetics  2015;7(1):1.
Background
The dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.
Results
We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).
Conclusions
ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13148-014-0036-2
PMCID: PMC4307187  PMID: 25628764
Ovarian cancer; Epigenetics; ABCA1
3.  Effects of the Endocrine-Disrupting Chemical DDT on Self-Renewal and Differentiation of Human Mesenchymal Stem Cells 
Background: Although the global use of the endocrine-disrupting chemical DDT has decreased, its persistence in the environment has resulted in continued human exposure. Accumulating evidence suggests that DDT exposure has long-term adverse effects on development, yet the impact on growth and differentiation of adult stem cells remains unclear.
Objectives: Human mesenchymal stem cells (MSCs) exposed to DDT were used to evaluate the impact on stem cell biology.
Methods: We assessed DDT-treated MSCs for self-renewal, proliferation, and differentiation potential. Whole genome RNA sequencing was performed to assess gene expression in DDT-treated MSCs.
Results: MSCs exposed to DDT formed fewer colonies, suggesting a reduction in self-renewal potential. DDT enhanced both adipogenic and osteogenic differentiation, which was confirmed by increased mRNA expression of glucose transporter type 4 (GLUT4), lipoprotein lipase (LpL), peroxisome proliferator-activated receptor gamma (PPARγ), leptin, osteonectin, core binding factor 1 (CBFA1), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Expression of factors in DDT-treated cells was similar to that in estrogen-treated MSCs, suggesting that DDT may function via the estrogen receptor (ER)-mediated pathway. The coadministration of ICI 182,780 blocked the effects of DDT. RNA sequencing revealed 121 genes and noncoding RNAs to be differentially expressed in DDT-treated MSCs compared with controls cells.
Conclusion: Human MSCs provide a powerful biological system to investigate and identify the molecular mechanisms underlying the effects of environmental agents on stem cells and human health. MSCs exposed to DDT demonstrated profound alterations in self-renewal, proliferation, differentiation, and gene expression, which may partially explain the homeostatic imbalance and increased cancer incidence among those exposed to long-term EDCs.
Citation: Strong AL, Shi Z, Strong MJ, Miller DF, Rusch DB, Buechlein AM, Flemington EK, McLachlan JA, Nephew KP, Burow ME, Bunnell BA. 2015. Effects of the endocrine-disrupting chemical DDT on self-renewal and differentiation of human mesenchymal stem cells. Environ Health Perspect 123:42–48; http://dx.doi.org/10.1289/ehp.1408188
doi:10.1289/ehp.1408188
PMCID: PMC4286277  PMID: 25014179
4.  A Mathematical Model of Bimodal Epigenetic Control of miR-193a in Ovarian Cancer Stem Cells 
PLoS ONE  2014;9(12):e116050.
Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.
doi:10.1371/journal.pone.0116050
PMCID: PMC4278842  PMID: 25545504
5.  Decitabine reactivated pathways in platinum resistant ovarian cancer 
Oncotarget  2014;5(11):3579-3589.
Combination therapy with decitabine, a DNMTi and carboplatin resensitized chemoresistant ovarian cancer (OC) to platinum inducing promising clinical activity. We investigated gene-expression profiles in tumor biopsies to identify decitabine-reactivated pathways associated with clinical response. Gene-expression profiling was performed using RNA from paired tumor biopsies before and 8 days after decitabine from 17 patients with platinum resistant OC. Bioinformatic analysis included unsupervised hierarchical-clustering, pathway and GSEA distinguishing profiles of “responders” (progression-free survival, PFS>6months) and “non-responders” (PFS<6months). Functional validation of selected results was performed in OC cells/tumors. Pre-treatment tumors from responders expressed genes associated with enhanced glycosphingolipid biosynthesis, translational misregulation, decreased ABC transporter expression, TGF-β signaling, and numerous metabolic pathways. Analysis of post-treatment biopsies from responders revealed overexpression of genes associated with reduced Hedgehog pathway signaling, reduced DNA repair/replication, and cancer-associated metabolism. GO and GSEA analyses revealed upregulation of genes associated with glycosaminoglycan binding, cell-matrix adhesion, and cell-substrate adhesion. Computational findings were substantiated by experimental validation of expression of key genes involved in two critical pathways affected by decitabine (TGF-β and Hh). Gene-expression profiling identified specific pathways altered by decitabine and associated with platinum-resensitization and clinical benefit in OC. Our data could influence patient stratification for future studies using epigenetic therapies.
PMCID: PMC4116504  PMID: 25003579
platinum resistant ovarian cancer; decitabine; gene expression; chemosensitization; DNA methylation; pathway analysis
6.  MiR-373 targeting of the Rab22a oncogene suppresses tumor invasion and metastasis in ovarian cancer 
Oncotarget  2014;5(23):12291-12303.
Metastasis is major cause of mortality in patients with ovarian cancer. MiR-373 has been shown to play pivotal roles in tumorigenesis and metastasis; however, a role for miR-373 in ovarian cancer has not been investigated. In this study, we show that the miR-373 expression is down-regulated in human epithelial ovarian cancer (EOC) and inversely correlated with clinical stage and histological grade. Ectopic overexpression of miR-373 in human EOC cells suppressed cell invasion in vitro and metastasis in vivo, and the epithelial–mesenchymal transition process. Silencing the expression of miR-373 resulted in an increased migration and invasion of EOC cells. Using integrated bioinformatics analysis, gene expression arrays, and luciferase assay, we identified Rab22a as a direct and functional target of miR-373 in EOC cells. Expression levels of miR-373 were inversely correlated with Rab22a protein levels in human EOC tissues. Rab22a knockdown inhibited invasion and migration of EOC cells, increased E-cadherin expression, and suppressed the expression of N-cadherin. Moreover, overexpression of Rab22a abrogated miR-373-induced invasion and migration of EOC cells. Taken together, these results demonstrate that miR-373 suppresses EOC invasion and metastasis by directly targeting Rab22a gene, a new potential therapeutic target in EOC.
PMCID: PMC4323008  PMID: 25460499
ovarian cancer; miR-373; Rab22a; invasion; metastasis
7.  Mg2+ Effect on Argonaute and RNA Duplex by Molecular Dynamics and Bioinformatics Implications 
PLoS ONE  2014;9(10):e109745.
RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg2+ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg2+] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg2+ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg2+ influx channel, TRPM7, and one miRNA’s (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg2+ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs.
doi:10.1371/journal.pone.0109745
PMCID: PMC4201462  PMID: 25330448
8.  microRNA regulation of mammalian target of rapamycin expression and activity controls estrogen receptor function and RAD001 sensitivity 
Molecular Cancer  2014;13(1):229.
Background
The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer.
Methods and results
Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway.
Conclusions
miRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-229) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-229
PMCID: PMC4203920  PMID: 25283550
miR-155; mTOR; breast cancer; miRNA; Estrogen receptor
9.  Epithelial Progeny of Estrogen-Exposed Breast Progenitor Cells Display a Cancer-like Methylome 
Cancer research  2008;68(6):1786-1796.
Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non–estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer.
doi:10.1158/0008-5472.CAN-07-5547
PMCID: PMC4172329  PMID: 18339859
10.  Amplification of Distant Estrogen Response Elements Deregulates Target Genes Associated with Tamoxifen Resistance in Breast Cancer 
Cancer cell  2013;24(2):197-212.
SUMMARY
A causal role of gene amplification in tumorigenesis is well-known, while amplification of DNA regulatory elements as an oncogenic driver remains unclear. In this study, we integrated next-generation sequencing approaches to map distant estrogen response elements (DEREs) that remotely control transcription of target genes through chromatin proximity. Two densely mapped DERE regions located on chromosomes 17q23 and 20q13 were frequently amplified in ERα-positive luminal breast cancer. These aberrantly amplified DEREs deregulated target gene expression potentially linked to cancer development and tamoxifen resistance. Progressive accumulation of DERE copies was observed in normal breast progenitor cells chronically exposed to estrogenic chemicals. These findings may extend to other DNA regulatory elements, the amplification of which can profoundly alter target transcriptome during tumorigenesis.
doi:10.1016/j.ccr.2013.07.007
PMCID: PMC3890247  PMID: 23948299
11.  Epigenetic Resensitization to Platinum in Ovarian Cancer 
Cancer research  2012;72(9):2197-2205.
Preclinical studies have shown that hypomethylating agents reverse platinum resistance in ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining study, we tested the clinical and biologic activity of low-dose decitabine administered before carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate (RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood mononuclear cells and tumors. The number of demethylated genes was greater (P < 0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244 genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included cytokine–cytokine receptor interactions, drug transporters, and mitogen-activated protein kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis (P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and HOXA11 in tumors positively correlated with PFS (P < 0.05). Together, the results of this study suggest that low-dose decitabine altered DNA methylation of genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resulting in a high RR and prolonged PFS.
doi:10.1158/0008-5472.CAN-11-3909
PMCID: PMC3700422  PMID: 22549947
12.  3D culture adds an extra dimension to targeted epigenetic therapies 
Cell Cycle  2013;12(14):2173-2174.
doi:10.4161/cc.25551
PMCID: PMC3755066  PMID: 23803725
ovarian cancer; epigenetics; EZH2; histone methyltransferase; 3D culture; H3K27; ECM
13.  A unique histone deacetylase inhibitor alters microRNA expression and signal transduction in chemoresistant ovarian cancer cells 
Cancer Biology & Therapy  2012;13(8):681-693.
Previously, we demonstrated potent antineoplastic activity of a distinctive histone deacetylase inhibitor (HDACI), AR42, against chemoresistant CP70 ovarian cancer cells in vitro and in vivo. Here, in follow-up to that work, we explored AR42 global mechanisms-of-action by examining drug-associated, genome-wide microRNA and mRNA expression profiles, which differed from those of the well-studied HDACI vorinostat. Expression of microRNA genes in negative correlation with their “target” coding gene (mRNA) transcripts, and transcription factor genes with expression positively correlated with coding genes having their cognate binding sites, were identified and subjected to gene ontology analyses. Those evaluations showed AR42 gene expression patterns to negatively correlate with Wnt signaling (> 18-fold induction of SFRP1), the epithelial-to-mesenchymal transition (40% decreased ATF1), and cell cycle progression (33-fold increased 14-3-3σ). By contrast, AR42 transcriptome alterations correlated positively with extrinsic (“death receptor”) apoptosis (> 2.3-fold upregulated DAPK) and favorable ovarian cancer histopathology and prognosis. Inhibition of Wnt signaling was experimentally validated by: (1) > 2.6-fold reduced Wnt reporter activity; and (2) 36% reduction in nuclear, activated β-catenin. Likely AR42 induction of multiple (type I or type II autophagic) cell death cascades was further supported by 57% decreased reliance upon reactive oxygen, increased mitochondrial membrane disruption, and caspase independence, as compared with vorinostat. Taken together, we demonstrate distinct antineoplastic pathway alterations, in aggressive ovarian cancer cells, following treatment with a promising HDACI, AR42. These combined computational and experimental approaches may also represent a straightforward means for mechanistic studies of other promising antineoplastics, and/or the identification of agents that may complement epigenetic therapies.
doi:10.4161/cbt.20086
PMCID: PMC3408973  PMID: 22549158
epithelial-to-mesenchymal transition; histone deacetylase inhibitor; microRNA; ovarian cancer; Wnt signaling
14.  Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers 
Epigenetics  2013;8(6):624-634.
Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating ?hit? during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan?Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.
doi:10.4161/epi.24816
PMCID: PMC3857342  PMID: 23774800
DNA methylation; Hedgehog pathway; ZIC1; ZIC4; ovarian cancer
15.  Definition of a FoxA1 Cistrome that is Crucial for G1-S Phase Cell-Cycle Transit in Castration-Resistant Prostate Cancer 
Cancer research  2011;71(21):6738-6748.
The enhancer pioneer transcription factor FoxA1 is a global mediator of steroid receptor (SR) action in hormone-dependent cancers. In castration-resistant prostate cancer (CRPC), FoxA1 acts as an androgen receptor co-factor to drive G2-M phase cell-cycle transit. Here we describe a mechanistically distinct SR-independent role for FoxA1 in driving G1-S phase cell-cycle transit in CRPC. By comparing FoxA1 binding sites in prostate cancer cell genomes, we defined a co-dependent set of FoxA1-MYBL2 and FoxA1-CREB1 binding sites within the regulatory regions of the Cyclin E2 and E2F1 genes that are critical for CRPC growth. Binding at these sites upregulate the Cyclin E2 and Cyclin A2 genes in CRPC but not in earlier stage androgen-dependent prostate cancer (ADPC), establishing a stage-specific role for this pathway in CRPC growth. Mechanistic investigations indicated that FoxA1, MYBL2 or CREB1 induction of histone H3 acetylation facilitated nucleosome disruption as the basis for co-dependent transcriptional activation and G1-S phase cell-cycle transit. Our findings establish FoxA1 as a pivotal driver of the cell-cycle in CRPC which promotes G1-S phase transit as well as G2-M phase transit through two distinct mechanisms.
doi:10.1158/0008-5472.CAN-11-1882
PMCID: PMC4081454  PMID: 21900400
FoxA1; cistrome; cell-cycle G1-S progression; castration-resistant prostate cancer
16.  The histone deacetylase inhibitor trichostatin A alters microRNA expression profiles in apoptosis-resistant breast cancer cells 
Oncology reports  2011;27(1):10-16.
The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.
doi:10.3892/or.2011.1488
PMCID: PMC3982613  PMID: 21971930
microRNA; trichostatin A; histone deacetylase; MCF-7; breast cancer; drug resistance
17.  Multivalent epigenetic marks confer microenvironment-responsive epigenetic plasticity to ovarian cancer cells 
Epigenetics  2010;5(8):716-729.
“Epigenetic plasticity” refers to the capability of mammalian cells to alter their differentiation status via chromatin remodeling-associated alterations in gene expression. While epigenetic plasticity has been best associated with lineage commitment of embryonic stem cells, recent studies have demonstrated chromatin remodeling even in terminally differentiated normal cells and advanced-stage melanoma and breast cancer cells, in context-dependent responses to alterations in their microenvironment. In the current study, we extend this attribute of epigenetic plasticity to aggressive ovarian cancer cells, by using an integrative approach to associate cellular phenotypes with chromatin modifications (“ChIP-chip”) and mRNA and microRNA expression. While we identified numerous gene promoters possessing the well-known “bivalent mark” of H3K27me3/H3K4me2, we also report 14 distinct, lesser known bi-, tri- and tetravalent combinations of activating and repressive chromatin modifications, in platinum-resistant CP 70 ovarian cancer cells. The vast majority (>90%) of all the histone marks studied localized to regions within 2,000 bp of transcription start sites, supporting a role in gene regulation. Upon a simple alteration in the microenvironment, transition from two- to three-dimensional culture, an increase (17–38%) in repressive-only marked promoters was observed, concomitant with a decrease (31–21%) in multivalent (i.e., juxtaposed permissive and repressive histone marked) promoters. Like embryonic/tissue stem and other (non-ovarian) carcinoma cells, ovarian cancer cell epigenetic plasticity reflects an inherent transcriptional flexibility for context-responsive alterations in phenotype. It is possible that this plasticity could be therapeutically exploited for the management of this lethal gynecologic malignancy.
doi:10.4161/epi.5.8.13014
PMCID: PMC3052886  PMID: 20676026
histone modifications; gene expression; chromatin remodeling; ovarian cancer; epigenetic plasticity; tumor microenvironment; bivalent histone mark
18.  Three-tiered role of the pioneer factor GATA2 in promoting androgen-dependent gene expression in prostate cancer 
Nucleic Acids Research  2014;42(6):3607-3622.
In prostate cancer, androgen receptor (AR) binding and androgen-responsive gene expression are defined by hormone-independent binding patterns of the pioneer factors FoxA1 and GATA2. Insufficient evidence of the mechanisms by which GATA2 contributes to this process precludes complete understanding of a key determinant of tissue-specific AR activity. Our observations suggest that GATA2 facilitates androgen-responsive gene expression by three distinct modes of action. By occupying novel binding sites within the AR gene locus, GATA2 positively regulates AR expression before and after androgen stimulation. Additionally, GATA2 engages AR target gene enhancers prior to hormone stimulation, producing an active and accessible chromatin environment via recruitment of the histone acetyltransferase p300. Finally, GATA2 functions in establishing and/or sustaining basal locus looping by recruiting the Mediator subunit MED1 in the absence of androgen. These mechanisms may contribute to the generally positive role of GATA2 in defining AR genome-wide binding patterns that determine androgen-responsive gene expression profiles. We also find that GATA2 and FoxA1 exhibit both independent and codependent co-occupancy of AR target gene enhancers. Identifying these determinants of AR transcriptional activity may provide a foundation for the development of future prostate cancer therapeutics that target pioneer factor function.
doi:10.1093/nar/gkt1382
PMCID: PMC3973339  PMID: 24423874
19.  Inhibition of MCL-1 enhances lapatinib toxicity and overcomes lapatinib resistance via BAK-dependent autophagy 
Cancer biology & therapy  2009;8(21):2084-2096.
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-Xl/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3–12 h.The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knockdown of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-Xl enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.
PMCID: PMC3887451  PMID: 19823038
lapatinib; obatoclax; autophagy; cell death; resistance
20.  A Phase I and Pharmacodynamic Study of Decitabine in Combination with Carboplatin in Patients with Recurrent, Platinum-Resistant, Epithelial Ovarian Cancer 
Cancer  2010;116(17):4043-4053.
Background:
Aberrant DNA methylation is a hallmark of cancer and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. Based on preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, we conducted a phase I trial of low dose decitabine combined with carboplatin, in patients with recurrent, platinum-resistant ovarian cancer.
Methods:
Decitabine was administered i.v. daily for five days, prior to carboplatin (AUC 5) on day 8 of a 28-day cycle. Using a standard 3+3 dose escalation decitabine was tested at two dose levels: 10 mg/m2 (seven patients) or 20 mg/m2 (three patients). Peripheral blood mononuclear cells (PBMCs) and plasma collected on days 1 (pre-treatment), 5, 8, and 15, were utilized to assess global (LINE-1 repetitive element) and gene-specific DNA methylation.
Results:
Dose-limiting toxicity (DLT) at the 20 mg/m2 dose was grade 4 neutropenia (2 patients) and no DLTs were observed at 10 mg/m2. Most common toxicities were nausea, allergic reactions, neutropenia, fatigue, anorexia, vomiting, and abdominal pain, the majority being grades 1-2. One complete response was observed, and three additional patients had stable disease for ≥ six months. LINE-1 hypomethylation on days 8 and 15 was detected in DNA from PBMCs. Of five ovarian cancer-associated methylated genes, HOXA11 and BRCA1 were demethylated in plasma on days 8 and 15.
Conclusions:
Repetitive low-dose decitabine is tolerated when combined with carboplatin in ovarian cancer patients, and demonstrates biological (i.e., DNA-hypomethylating) activity justifying further testing for clinical efficacy.
doi:10.1002/cncr.25204
PMCID: PMC2930033  PMID: 20564122
ovarian cancer; decitabine; epigenetic biomarkers; chemosensitization; platinum resistance
21.  Epigenetic Therapies for Chemoresensitization of Epithelial Ovarian Cancer 
Gynecologic oncology  2009;116(2):195.
Summary
Epigenetic drugs have been shown to enhance gene expression and drug sensitivity in ovarian cancer cell lines and animal models. Based on promising pre-clinical studies, DNA methylation inhibitors in combination with existing chemotherapeutic agents have the potential for overcoming acquired drug resistance, laying the foundation for this specific class of epigenetic drug in ovarian cancer clinical trials. The recent completion of phase I trials of decitabine have yielded important information on dosing schedules and biological endpoints for evaluating patient responses. In addition, epigenetic drug effects on pharmacodyamic targets are beginning to emerge, and predictive epigenetic biomarkers and next generation epigenome therapeutics are being developed for application in clinical settings for ovarian cancer patients.
doi:10.1016/j.ygyno.2009.09.043
PMCID: PMC2813995  PMID: 19854495
22.  MEK5/ERK5 Signaling Suppresses Estrogen Receptor Expression and Promotes Hormone-Independent Tumorigenesis 
PLoS ONE  2013;8(8):e69291.
Endocrine resistance and metastatic progression are primary causes of treatment failure in breast cancer. While mitogen activated protein kinases (MAPKs) are known to promote ligand-independent cell growth, the role of the MEK5-ERK5 pathway in the progression of clinical breast carcinoma remains poorly understood. Here, we demonstrated increased ERK5 activation in 30 of 39 (76.9%) clinical tumor samples, as well as across breast cancer cell systems. Overexpression of MEK5 in MCF-7 cells promoted both hormone-dependent and hormone-independent tumorigenesis in vitro and in vivo and conferred endocrine therapy resistance to previously sensitive breast cancer cells. Expression of MEK5 suppressed estrogen receptor (ER)α, but not ER-β protein levels, and abrogated downstream estrogen response element (ERE) transcriptional activity and ER-mediated gene transcription. Global gene expression changes associated with upregulation of MEK5 included increased activation of ER-α independent growth signaling pathways and promotion of epithelial-to-mesenchymal transition (EMT) markers. Taken together, our findings show that the MEK5-ERK5 pathway mediates progression to an ER(−), mesenchymal and endocrine therapy resistant phenotype. Given the need for new clinical therapeutic targets, our results demonstrate the therapeutic potential of targeting the MEK5-ERK5 pathway in breast cancer.
doi:10.1371/journal.pone.0069291
PMCID: PMC3739787  PMID: 23950888
23.  Integrated analysis of genome-wide DNA methylation and gene expression profiles in molecular subtypes of breast cancer 
Nucleic Acids Research  2013;41(18):8464-8474.
Aberrant DNA methylation of CpG islands, CpG island shores and first exons is known to play a key role in the altered gene expression patterns in all human cancers. To date, a systematic study on the effect of DNA methylation on gene expression using high resolution data has not been reported. In this study, we conducted an integrated analysis of MethylCap-sequencing data and Affymetrix gene expression microarray data for 30 breast cancer cell lines representing different breast tumor phenotypes. As well-developed methods for the integrated analysis do not currently exist, we created a series of four different analysis methods. On the computational side, our goal is to develop methylome data analysis protocols for the integrated analysis of DNA methylation and gene expression data on the genome scale. On the cancer biology side, we present comprehensive genome-wide methylome analysis results for differentially methylated regions and their potential effect on gene expression in 30 breast cancer cell lines representing three molecular phenotypes, luminal, basal A and basal B. Our integrated analysis demonstrates that methylation status of different genomic regions may play a key role in establishing transcriptional patterns in molecular subtypes of human breast cancer.
doi:10.1093/nar/gkt643
PMCID: PMC3794600  PMID: 23887935
24.  Apigenin Inhibits Antiestrogen-resistant Breast Cancer Cell Growth through Estrogen Receptor-α-dependent and -independent Mechanisms 
Molecular cancer therapeutics  2008;7(7):2096-2108.
Breast cancer resistance to the antiestrogens tamoxifen and fulvestrant is accompanied by alterations in both estrogen-dependent and -independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either tamoxifen or fulvestrant. We found that apigenin can function as both an estrogen and antiestrogen, in a dose-dependent manner. At low concentrations (1μM), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (≥10μM), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either tamoxifen or fulvestrant demonstrated synergistic, growth-inhibitory effects on both antiestrogen-sensitive and -resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or antiestrogen, effects of the drug on estrogen receptor-α (ERα) transactivation activity, mobility, stability, and ERα-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERα and its co-activator AIB1 (amplified in breast cancer-1). However, higher doses (> 10μM) of apigenin inhibited ERα mobility (as determined by FRAP assays), downregulated ERα and AIB1 expression levels, and inhibited multiple protein kinases, including p38, PKA, MAPK and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERα-dependent and -independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer.
doi:10.1158/1535-7163.MCT-07-2350
PMCID: PMC2559959  PMID: 18645020
apigenin; antiestrogen; fulvestrant; tamoxifen; breast cancer
25.  MicroRNA and mRNA integrated analysis (MMIA): a web tool for examining biological functions of microRNA expression 
Nucleic Acids Research  2009;37(Web Server issue):W356-W362.
MicroRNAs (miRNAs) are small (19–24 nt), nonprotein-coding nucleic acids that regulate specific ‘target’ gene products via hybridization to mRNA transcripts, resulting in translational blockade or transcript degradation. Although miRNAs have been implicated in numerous developmental and adult diseases, their specific impact on biological pathways and cellular phenotypes, in addition to miRNA gene promoter regulation, remain largely unknown. To improve and facilitate research of miRNA functions and regulation, we have developed MMIA (microRNA and mRNA integrated analysis), a versatile and user-friendly web server. By incorporating three commonly used and accurate miRNA prediction algorithms, TargetScan, PITA and PicTar, MMIA integrates miRNA and mRNA expression data with predicted miRNA target information for analyzing miRNA-associated phenotypes and biological functions by gene set analysis, in addition to analysis of miRNA primary transcript gene promoters. To assign biological relevance to the integrated miRNA/mRNA profiles, MMIA uses exhaustive human genome coverage, including classification into various disease-associated genes as well as conventional canonical pathways and Gene Ontology. In summary, this novel web server (cancer.informatics.indiana.edu/mmia) will provide life science researchers with a valuable tool for the study of the biological (and pathological) causes and effects of the expression of this class of interesting protein regulators.
doi:10.1093/nar/gkp294
PMCID: PMC2703907  PMID: 19420067

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